Streptomyces griseoaurantiacus HMQAUfx21024, biocontrol inoculant and preparation method and application of biocontrol inoculant

Abstract

The invention relates to the field of biocontrol of plant diseases. The invention provides a Streptomyces griseoaurantiacus strain HMQAUfx21024, the strain is preserved in the China General Microbiological Culture Collection Center on January 3, 2025, and the preservation registration number of the strain is CGMCC No. 33290. The invention further provides a preparation method of the strain HMQAUfx21024, and the strain HMQAUfx21024 has the advantages that the strain HMQAUfx21024 can be used for preparing the strain HMQAUfx21024; the invention also provides a biocontrol inoculant. The invention also provides a preparation method of the biocontrol inoculant. The invention also provides application of the strain HMQAUfx21024 or the biocontrol microbial inoculum as described above or the biocontrol microbial inoculum prepared by the preparation method as described above. The invention further provides a method for preventing and treating plant diseases. The biocontrol agent is specially developed for cucumber downy mildew and target leaf spot, due to the fact that the biocontrol agent is a biological agent, a series of problems caused by use of chemical pesticides are completely avoided, pollution-free production of greenhouse vegetables is facilitated, farmers can not use or reduce the use amount of other chemical pesticides, expenses can be saved for the farmers, and the cost is reduced. Vegetable export is facilitated, and the income of farmers can be increased.

Description

Technical Field

[0001] The invention belongs to the field of plant disease biological control, and specifically relates to a Streptomyces griseoaurantifolia HMQAUfx21024 and an application thereof. Background Art

[0002] cucumber( Cucumis sativus Linn. ), belongs to the Cucurbitaceae ( Cucurbitaceae ) Cucumber Cucumber ), originated in the tropical rainforest area on the southern foot of the Himalayas, is an annual climbing herb with a cultivation history of more than 2,000 years in my country (Li Huaizhi, 2003). It has now become one of the top ten cultivated vegetable crops in the world. In 2020, the global cucumber planting area was about 2.25 million hectares.

[0003] Cucumber downy mildew is caused by Pseudocerona cubaniensis ( Pseudoperonospora cubensis ) is an important leaf disease caused by frostbite (Thomas, 1986), and is also a devastating disease on cucumbers in protected areas, seriously affecting the yield and quality of cucumbers (Fu Shuyun, 1983; Shi Yanxia, ​​2002). There are four main measures for the prevention and control of cucumber downy mildew: disease-resistant varieties are time-consuming and labor-intensive and have slow results; ecological control is difficult to control environmental conditions; chemical pesticides are prone to produce pesticide residues, which have adverse effects on human health and pollute the environment; and biological control is environmentally friendly, efficient, and pollution-free, becoming a new measure for the prevention and control of cucumber downy mildew.

[0004] Cucumber target spot is caused by Coryneformis spores ( Corynespora cassicola ) is a leaf disease caused by the disease. It first occurred in Jilin Province in the early 1960s, in Liaoning and Hebei in the 1990s, and in Henan, Shandong, Gansu, Ningxia, Beijing and Shanghai in the 21st century. In protected areas, the incidence of cucumber target spot is generally 20-30%, and in severe cases it can reach 60-70%, causing a large number of leaves to necrotize and fall off, and a significant reduction in yield or even a total loss of yield.

[0005] However, the existing technology lacks biocontrol agents with high efficacy against cucumber downy mildew, cucumber target spot and other plant diseases. How to screen out a suitable actinomycete for preparing biocontrol agents is a technical problem that needs to be solved in the field of plant disease biocontrol. Summary of the invention

[0006] The present invention aims to address the deficiencies of the prior art and provides a Streptomyces griseoaurantifolia HMQAUfx21024 and an application thereof.

[0007] The first object of the present invention is to provide a biocontrol actinomycete isolated from spinach rhizosphere soil, Streptomyces griseo-orange ( Streptomyces griseoaurantiacus) strain HMQAUfx21024, enriching the strain resources of plant disease biocontrol bacteria and laying the foundation for the research and development of biocontrol bacteria.

[0008] The second object of the present invention is to provide a biocontrol agent.

[0009] The third object of the present invention is to provide a method for preparing the above-mentioned spore suspension liquid, fermentation liquid and fermentation filtrate biocontrol agents.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned Streptomyces griseoaurantifolia strain HMQAUfx21024 or the above-mentioned biocontrol agent in inhibiting plant pathogens, preparing products for inhibiting plant pathogens, preventing and controlling diseases caused by plant pathogens, and preparing products for preventing and controlling diseases caused by plant pathogens.

[0011] A fifth object of the present invention is to provide use of the Streptomyces griseoaurantifolia strain HMQAUfx21024 or the biocontrol agent described above in plant disease control.

[0012] A sixth object of the present invention is to provide a method for preventing and controlling plant diseases.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions: A strain of Streptomyces griseo-orange Streptomyces griseoaurantiacus ) strain HMQAUfx21024 was deposited in the General Microbiology Center of China Microbiological Culture Collection on January 3, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit registration number: CGMCC No. 33290.

[0014] Based on the same inventive concept, the present invention provides a biocontrol agent, wherein the active ingredient of the biocontrol agent includes the bacterial suspension, fermentation liquid or fermentation filtrate of the Streptomyces griseoaurantifolia strain HMQAUfx21024 as described above.

[0015] The biocontrol agent as described above, wherein the total viable bacterial concentration of Streptomyces griseo-orange strain HMQAUfx21024 in the biocontrol agent is 1×10 5 ~2×10 5 cfu / mL.

[0016] Optionally, the present invention provides a method for preparing a biocontrol agent, the specific steps comprising: The Streptomyces griseo-orange strain HMQAUfx21024 was activated on Gao's No. 1 solid culture medium at 25-35°C for 3-5 days, and then sterile water was added to prepare a spore suspension to obtain a biocontrol agent.

[0017] Optionally, the present invention provides a method for preparing a biocontrol agent, the specific steps comprising: S1. Cultivation of seed solution: After activating the Streptomyces griseo-orange strain HMQAUfx21024 on Gao's solid medium No. 1 at 25-35°C for 3-5 days, 3-5 bacterial cakes were taken from the edge of the colony and inoculated into Gao's liquid medium No. 1 at 25-35°C and 150-200 rpm for 3-5 days to obtain seed solution; S2. Preparation of fermentation broth: The seed solution obtained in step S1 was added to a conical flask containing a certain amount of basic fermentation medium at a ratio of 10 to 15% of the inoculum, and cultured at 25 to 35°C and 150 to 220 rpm for 3 to 5 days to prepare a fermentation broth to obtain a biocontrol agent.

[0018] Optionally, the present invention provides a method for preparing a biocontrol agent, the specific steps comprising: S1. Cultivation of seed solution: After activating the Streptomyces griseo-orange strain HMQAUfx21024 on Gao's solid medium No. 1 at 25-35°C for 3-5 days, 3-5 bacterial cakes were taken from the edge of the colony and inoculated into Gao's liquid medium No. 1 for 3-5 days at 25-35°C and 150-200 rpm for shaking culture to obtain seed solution; S2. Preparation of fermentation broth: The seed solution obtained in step S1 was added to a certain amount of the base fermentation medium in an inoculum ratio of 10 to 15%, and the fermentation broth was prepared by shaking and culturing at 25 to 35 ° C and 150 to 220 rpm for 3 to 5 days; S3. Preparation of fermentation filtrate: The fermentation broth obtained from step S2 is centrifuged at 4-6°C, 6000-9000rpm for 20-30min, pH 7-8, and filtered through a microporous membrane to prepare a fermentation filtrate, that is, a biocontrol agent.

[0019] According to the preparation method of the biocontrol agent as described above, the components and contents of the Gao's No. 1 solid culture medium in step S1 are as follows: 20g soluble starch, 0.5g sodium chloride, 1g potassium nitrate, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferric sulfate, 20g agar, and 1000mL distilled water; the components and contents of the Gao's No. 1 liquid culture medium in step S1 are as follows: 20g soluble starch, 0.5g sodium chloride, 1g potassium nitrate, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferric sulfate, and 1000mL distilled water; the basic fermentation medium in step S2 is millet medium, and the components and contents are as follows: 10.0g millet, 10.0g glucose, 3.0g peptone, 2.5g sodium chloride, 2.0g calcium carbonate, 1000mL distilled water, pH7.2-7.4.

[0020] Based on the same inventive concept, the present invention also provides a use of the Streptomyces griseo-orange strain HMQAUfx21024 as described above, or the biocontrol agent as described above, or the biocontrol agent prepared by the preparation method as described above in the following (a1) or (a2) or (a3) ​​or (a4), (a1) inhibiting plant pathogens; (a2) preparing a product for inhibiting plant pathogens; (a3) ​​preventing and controlling diseases caused by plant pathogens; (a4) preparing a product for preventing and controlling diseases caused by plant pathogens; the plant pathogen is Pseudocytin ( Pseudoperonospora cubensis )、Spores of sphaerocephala ( Corynespora cassicola )、Mucor circinelloides( Mucor circinelloides )、Forest-dwelling Pythium Pythium forest )、Botrytis cinerea( Botryosphaeria dothidea )、False cocoa hair color two cells ( Lasiodiplodia pseudotheobroma )、Pythium terrestris( Pythium irregulare ), Sclerotinia sclerotiorum ( Sclerotinia sclerotia )、Botrytis cinerea( Botrytis cinerea )、Phomopsis sphaeroides( Phomopsis sp.), Pseudodiscus hirsutus ( Pestalotiopsis sp.), Paper-like spores ( Paper mulberry )、Stoma betae Beetroot soup )、Fusarium solani( Fusarium solani ), Pseudofusorium graminearum ( Fusarium pseudogramineum ), Fusarium oxysporum ( Fusarium oxysporum )、Siamese Anthrax ( Colletotrichum siamense )、White rot spores( Little rabbit diplodia )、Stolonifera( Stemphylium botryosum )、Phytophthora capsici( Phytophthora capsicum )、Pythium glomerulosa( Pythium myriotylum )、Rhizoctonia solani( Rhizoctonia solani )、Clondrosporium fulva Cladosporium yellow ) Alternaria solani )、Pythium aphanidermatum( Pythium aphanidermatum )、Collegiosporium Colletorichum gloeosporioides )、Pythium spinulosum( Pythium spinosum )、Circular Sporangium ( Colletotrichum orbiculare ).

[0021] Based on the same inventive concept, the present invention also provides an application of the gray-orange Streptomyces strain HMQAUfx21024 as described above, or the biocontrol agent as described above, or the biocontrol agent prepared by the preparation method as described above in plant disease prevention and control, wherein the plant disease is any one of cucumber downy mildew, cucumber target spot disease, lily gray mold, bean gray mold, celery sclerotinia, Chinese cabbage sclerotinia, grape white rot, blueberry hairy two-spora branch blight, spinach rot, lettuce leaf spot, spinach Fusarium root rot, blueberry leaf spot, quinoa branch blight, garlic dry rot, kiwi soft rot, wheat stem base rot, pepper blight, ginger root rot, cabbage Rhizoctonia root rot, tomato leaf mold, eggplant early blight, cucumber cotton rot, strawberry anthracnose, cucumber rot, cucumber wilt, watermelon wilt, and cucumber anthracnose.

[0022] Based on the same inventive concept, the present invention also provides a method for preventing and controlling plant diseases. When preventing and controlling plant diseases, the gray-orange Streptomyces strain HMQAUfx21024 as described above or the biocontrol agent as described above or the biocontrol agent prepared by the preparation method as described above is used for coating, dipping, spraying, atomizing, irrigating, powdering, broadcasting, foaming, applying or spraying on the disease-prone parts of the plant.

[0023] Compared with the existing technology, the effects and advantages of the present invention are: (1) The fermentation filtrate of Streptomyces griseo-orange strain HMQAUfx21024 provided by the present invention has a broad spectrum of antagonistic activity against plant pathogens, for example, it has different degrees of inhibitory effect on various pathogens such as Pseudocytin, ... In particular, it has a significant inhibitory effect on Mucor circinelloides, Pythium arvense, Botrytis cinerea, and Pseudococcidioides sclerotiorum, with an inhibition rate of more than 95%; followed by Pythium sclerotiorum, Sclerotinia sclerotiorum, Botrytis cinerea, Phomopsis sclerotiorum, Pseudococcidioides sclerotiorum, and Pseudococcidioides sclerotiorum, with inhibition rates all above 75%. Experiments show that the griseo-orange Streptomyces strain HMQAUfx21024 has a broad antibacterial spectrum, good antibacterial effect, and high biocontrol potential.

[0024] (2) The Streptomyces griseus strain HMQAUfx21024 provided by the present invention has different degrees of inhibitory effects on various pathogens. In particular, the inhibition rate of Phytophthora capsici reached 91.43%, followed by Botrytis cinerea and Pythium glomerulosa, with inhibition rates of more than 85%, indicating that the Streptomyces griseus strain HMQAUfx21024 also has a good antibacterial effect.

[0025] (3) The gray-orange Streptomyces strain HMQAUfx21024 provided by the present invention is effective against cucumber downy mildew, cucumber target spot disease, lily gray mold, bean gray mold, celery sclerotinia, Chinese cabbage sclerotinia, grape white rot, blueberry hairy dispora branch blight, spinach rot, lettuce leaf spot, spinach Fusarium root rot, blueberry leaf spot, quinoa branch blight, garlic dry rot, kiwifruit soft rot, wheat stem rot, pepper blight, ginger root rot, cabbage Rhizoctonia root rot, tomato leaf mold, eggplant early blight, cucumber cotton rot Diseases of the plant species such as scab, strawberry anthracnose, cucumber rot, cucumber wilt, watermelon wilt, and cucumber anthracnose have been shown to be effective in preventing and controlling plant diseases. This indicates that the application of this strain, on the one hand, helps to reduce the use of chemical pesticides and reduce environmental pollution, and on the other hand, can develop biological agents for diseases of various crops such as cucumber, lily, kidney bean, celery, cabbage, grape, blueberry, spinach, lettuce, quinoa, garlic, kiwi, wheat, pepper, ginger, cabbage, tomato, eggplant, strawberry, and watermelon, so as to improve the yield and quality of crops.

[0026] (4) The present invention is a biological control agent specially developed for cucumber downy mildew and target spot disease. Since it is a biological agent, it is completely free of the series of problems caused by the use of chemical pesticides. It is therefore conducive to the pollution-free production of greenhouse vegetables. Farmers do not need to use or reduce the use of other chemical pesticides. This not only saves farmers money, but also facilitates the export of vegetables and increases farmers' income. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of rescreening actinomycete strains of cucumber downy mildew; Figure 2 The results of rescreening of antagonistic actinomycete strains against cucumber target spot disease; AN: photos of detached cucumber leaves; AG is the front side of the leaves, and HN is the back side of the leaves; A, H: control; B, I: HMQAUfx21024; C, J: HMQAUfx21061; D, K: HMQAUfx21007; E, L: HMQAUfx21060; F, M: HMQAUfx21025; G, N: HMQAUfx21070; Figure 3 The mycelial morphology of strain HMQAUfx21024; A: insert culture; B: spore mycelium; C: spore mycelium and spores; Figure 4 The culture characteristics of strain HMQAUfx21024; A: front culture characteristics; B: back culture characteristics; C: single colony; Figure 5The culture characteristics of strain HMQAUfx21024 on different culture media; A: yeast extract maltose agar (ISP2); B: oatmeal agar (ISP3); C: inorganic salt starch (ISP4); D: glycerol aspartate agar (ISP5); E: Gao's agar No. 1; F: potato dextrose agar (PDA); Figure 6 This is the phylogenetic tree of strain HMQAUfx21024 constructed based on 16S rDNA sequence; the values ​​on the branch points are support rates; the scale 0.10 is the evolutionary distance; Figure 7 Plate inhibition effect of strain HMQAUfx21024 on pathogens. DETAILED DESCRIPTION

[0028] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] In the present invention, the Streptomyces griseoaurantifolia strain HMQAUfx21024 is referred to as "strain HMQAUfx21024" or "HMQAUfx21024" for short.

[0033] Example 1 Isolation and screening of biocontrol actinomycete strains 1.1 Isolation of actinomycete strains In September 2021, spinach rhizosphere soil was collected from Liangjiahuang, Jimo City, Qingdao City, Shandong Province. The actinomycete strains were isolated and purified by the dilution coating method and plate streak method, respectively, and stored in 30% glycerol.

[0034] The preparation method of the bacterial cell preservation solution is as follows: glycerol is diluted with 0.9% physiological saline to a concentration of 30% to obtain the bacterial cell preservation solution.

[0035] 1.2 Screening of biocontrol actinomycete strains 1.2.1 Host plants and pathogens tested Cucumber variety: Improved Lu cucumber No. 3.

[0036] Collection, preservation, propagation and preparation of sporangium suspension of cucumber downy mildew: In March 2022, cucumber downy mildew diseased leaves were collected in Liangjiahuang, Jimo City, Qingdao City, Shandong Province. The detached leaves of the improved Lu cucumber No. 3 were used to preserve and propagate cucumber downy mildew. After a fresh mold layer grew on the back of the detached cucumber leaves, the sporangium suspension was prepared into a concentration of 2×10 5 cfu / mL and 10 5 Two concentrations of cfu / mL are available for use.

[0037] 1.2.2 Preparation of actinomycete fermentation filtrate Cultivation of seed solution: After 60 actinomycetes, including strain HMQAUfx21024, were activated on Gao's No. 1 solid medium at 28°C for 3 days, three 5mm diameter bacterial cakes were inoculated into Gao's No. 1 liquid medium for shaking culture at 28°C and 180rpm for 3 days to obtain seed solution. The components and contents of Gao's No. 1 solid medium are as follows: soluble starch 20g, sodium chloride 0.5g, potassium nitrate 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.5g, iron sulfate 0.01g, agar 20g, distilled water 1000mL. The components and contents of Gao's No. 1 liquid culture medium are as follows: soluble starch 20g, sodium chloride 0.5g, potassium nitrate 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.5g, iron sulfate 0.01g, distilled water 1000mL.

[0038] Preparation of fermentation broth: Add the seed liquid at a ratio of 10% to a 150 mL conical flask containing 80 mL of basic fermentation medium, and culture at 28 ° C and 180 rpm for 2.5 days to prepare the fermentation broth; the basic fermentation medium is millet medium, and the ingredients and content are as follows: 10.0 g millet, 10.0 g glucose, 3.0 g peptone, 2.5 g sodium chloride, 2.0 g calcium carbonate, 1000 mL distilled water, pH 7.2-7.4.

[0039] Preparation of fermentation filtrate: The fermentation broth was centrifuged at 4°C, 8000 rpm for 20 min, pH 7.5, and filtered through a 0.22 μm microporous membrane to prepare sterile fermentation filtrate of each actinomycete strain.

[0040] 1.2.3 Screening of actinomycete strains The concave slide method was used to test the effects of 60 actinomycete fermentation filtrates on the sporangium of cucumber downy mildew (2×10 5 cfu / mL) release. The actinomycete fermentation filtrate was mixed with the sporangium suspension of cucumber downy mildew in a ratio of 1:1, and sterile water and sporangium suspension were mixed in a ratio of 1:1 as a blank control. The subsequent test steps refer to the method of Yan Lei (Yan Lei, 2013). Three replicates were set for each treatment, and the test was repeated twice.

[0041] The secondary screening used the detached leaf spray method to determine the inhibitory effect of the fermentation filtrate of the actinomycete strain selected after the primary screening on cucumber downy mildew. Select cucumber leaves of the same size, rinse them with tap water, absorb the water, and place them on a petri dish with wet filter paper with the back facing up. Spray the actinomycete fermentation filtrate evenly on the back of the leaves until it runs off. After the fermentation filtrate is slightly dry, inoculate the pathogen (10 5 cfu / mL of sporangium suspension); sterile water treatment as blank control. The inoculation was carried out by the detached leaf fixed point inoculation method, with 5 points on each leaf and 10μL of sporangium suspension on each point. After culturing at 20℃ in full darkness for 24h, the lesion size was measured after 7 days of alternating light and dark at 22℃ during the day and 18℃ at night. The disease index and control effect were calculated based on the lesion size. The grading standard for inoculation of detached leaves is shown in Table 1 (Fu Shuyun, 1984).

[0042] Table 1 Grading standards for cucumber downy mildew inoculated with detached leaves

[0043] Disease index = [∑(representative grade value × number of lesions) / (total number of lesions surveyed × highest grade value)] × 100 Relative prevention effect (%) = [(control disease index - treatment disease index) / control disease index] × 100% 1.3 Results of initial screening of biocontrol actinomycete strains The 60 actinomycetes isolated in the laboratory earlier were initially screened by the concave slide method, and sterile water treatment was used as a blank control. The test results showed that 11 strains of actinomycetes had a relative control effect of >60% on cucumber downy mildew, of which 4 strains had a relative control effect of 100% on cucumber downy mildew, namely HMQAUfx21007, HMQAUfx21024, HMQAUfx21033, and HMQAUfx21072; followed by 4 strains of actinomycetes with a relative control effect of 90.96~80.28% on cucumber downy mildew, namely HMQAUfx21044, HMQAUfx21061, HMQAUfx21060, HMQAUfx21025, and HMQAUfx21070; the remaining 3 strains of actinomycetes had a relative control effect of 66.23~61.74% on cucumber downy mildew, namely HMQAUfx21037, HMQAUfx21073, and HMQAUfx21049. The analysis of the significance of the relative protective effects showed that there were significant differences in the inhibition rates of sporangium release of cucumber downy mildew among strains HMQAUfx21007, HMQAUfx21024, HMQAUfx21033, HMQAUfx21072 and strain HMQAUfx21060 and strains below their relative protective effects (Table 2).

[0044] Table 2 Results of preliminary screening of actinomycete strains of cucumber downy mildew

[0045] Note: The letters after the numbers in the same column indicate P The difference was significant at the level of <0.05, the same below.

[0046] 1.4 Results of rescreening of biocontrol actinomycete strains The 9 actinomycetes with a relative protection efficiency of >80% after the initial screening were rescreened by the detached leaf spray method, and sterile water treatment was used as a blank control. The results showed that the strain HMQAUfx21024 had a strong inhibitory effect on cucumber downy mildew, with a relative protection efficiency of 91.37%. Figure 1 And as shown in Table 3.

[0047] Table 3 Results of rescreening of actinomycete strains of cucumber downy mildew

[0048] Example 2 Screening of biocontrol actinomycetes against cucumber target spot pathogen 2.1 Materials and Methods Pathogen: Cucumber spot pathogen: Coryneformis spp. Corynespora cassicola HMQAU200087 and prepared with sterile water to a concentration of 1 × 10 5 cfu / mL of spore suspension for later use.

[0049] Tested actinomycetes: 6 strains with good control effect on cucumber downy mildew (HMQAUfx21024, HMQAUfx21061, HMQAUfx21007, HMQAUfx21060, HMQAUfx21025, HMQAUfx21070).

[0050] Preparation of actinomycete fermentation filtrate: same as “1.2.2 Preparation of actinomycete fermentation broth”.

[0051] Preparation of fermentation broth of actinomycete strain HMQAUfx21024: Cultivation of seed liquid is the same as in “1.2.2 Preparation of fermentation filtrate of actinomycete strain”; preparation of fermentation broth is the same as in “1.2.2 Preparation of fermentation filtrate of actinomycete strain”.

[0052] Preparation of spore suspension of actinomycete strain HMQAUfx21024: After activating the spore suspension of Streptomyces griseo-orange strain HMQAUfx21024 on a 9-cm-diameter plate with 10 mL of Gao's solid medium No. 1 at 28°C for 3 days, 10 mL of sterile water was added to prepare a spore suspension of 1×10 5 cfu / mL spore suspension.

[0053] Initial screening of biocontrol actinomycetes against cucumber target spot pathogen: The filtrate medium method was used for initial screening. The actinomycete fermentation filtrate and PDA medium were mixed at a volume ratio of 1:10 to prepare the medium. The pathogen was inoculated with the lawn side facing down in the center of a plate (9 cm × 9 cm in size) and cultured at 25°C. When the control colony approached the edge of the plate, the colony radius was measured. The PDA plate without actinomycete fermentation filtrate was used as a blank control to calculate the inhibition rate.

[0054] Inhibition rate = [(colony radius of control group - colony radius of treatment group) / colony radius of control group] × 100%.

[0055] Rescreening of biocontrol actinomycetes against cucumber target spot disease: The inhibitory effect of the fermentation filtrate of the biocontrol strain on cucumber target spot disease was determined by the detached leaf spray method.

[0056] Select cucumber leaves of the same size, rinse them with tap water, dry them, and place them face up in a petri dish lined with wet filter paper for later use. Spray the actinomycete fermentation filtrate obtained in the initial screening evenly on the front of the leaves until it runs off. After the fermentation filtrate is slightly dry, inoculate the pathogen (1×10 5 cfu / mL of sporangium suspension); sterile water treatment as blank control. Each treatment was repeated 3 times. Placed in a constant temperature and moisturizing culture at 25℃, the disease degree of the leaves was observed after 7 days, the size of the lesions was measured, and the disease index and control effect were calculated based on the size of the lesions. The grading standard for inoculation of detached leaves is as follows (Weng Zuxin, 1991).

[0057] In vitro inhibitory effects of spore suspension and fermentation broth of strain HMQAUfx21024 on cucumber target spot disease: Select cucumber leaves of the same size, rinse them with tap water, dry them, and place them face up in a petri dish lined with wet filter paper for later use. 5 cfu / mL) and fermentation liquid were sprayed evenly on the front of the leaves until they were drained. After that, the cucumber leaves treated with spore suspension were cultured in an incubator at 28°C for 12 h of light for 24 h and then inoculated with pathogens (1×10 5 cfu / mL of sporangium suspension); cucumber leaves treated with fermentation liquid were inoculated with pathogens (1×10 5 cfu / mL of sporangium suspension); sterile water treatment as blank control. Each treatment was repeated 3 times. Placed in a constant temperature and moisturizing culture at 25℃, the disease degree of the leaves was observed after 7 days, the size of the lesions was measured, and the disease index and control effect were calculated based on the size of the lesions. The grading standard for inoculation of detached leaves is as follows (Weng Zuxin, 1991).

[0058] Table 4 Grading standards for cucumber target spot disease inoculated on detached leaves

[0059] Disease index = Σ (number of diseased plants at each level × representative value at each level) × 100 / (total number of plants surveyed × highest representative value) Control effect = [(control disease index - treatment disease index) / control disease index] × 100% 2.2 Screening results of biocontrol actinomycetes against cucumber target leaf spot pathogen The filtrate medium method was used for initial screening, and it was found that HMQAUfx21024 had the highest inhibition rate against cucumber target spot pathogen, which was 90.10%. The detached leaf spray method was used for rescreening, and it was found that the fermentation filtrate of the strain had a control effect of 92.00% against cucumber target spot pathogen. Figure 2 shown.

[0060] 2.3 In vitro efficacy of spore suspension and fermentation broth of strain HMQAUfx21024 against cucumber target spot disease The spore suspension of strain HMQAUfx21024 had a control effect of 91.6% on target spot disease on detached cucumber leaves. The fermentation liquid of strain HMQAUfx21024 had a control effect of 93.7% on target spot disease on detached cucumber leaves.

[0061] Example 3 Identification of biocontrol actinomycetes 3.1 Materials and methods 3.1.1 Test strains: The strain HMQAUfx21024 was maintained by the Fungal Laboratory of the College of Plant Medicine, Qingdao Agricultural University.

[0062] 3.1.2 Test culture medium: The culture medium (Table 5) refers to "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng and Huang Ying, 2010).

[0063] Table 5 Test culture medium and its use

[0064] 3.1.3 Observation of morphology and culture characteristics Morphological characteristics: The strain HMQAUfx21024 was cultured at 28°C using the insert method (Jiang Chaorui and Ruan Jisheng, 1982). After 3d, 5d, 7d, 10d, 14d and 21d, the coverslips inserted in the culture medium were taken out and the hyphae, aerial hyphae, spore hyphae and spores were observed under an optical microscope and photographed.

[0065] Culture characteristics: refer to "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng and Huang Ying, 2010). The strain HMQAUfx21024 was inoculated into 6 international common culture media (Table 6), and the growth and culture characteristics of the strain were observed under 28℃ culture conditions, and the aerial filaments, basal filaments and soluble pigments (culture medium color) were recorded.

[0066] 3.1.4 Physiological and biochemical identification Physiological and biochemical characteristics: The strain HMQAUfx21024 was inoculated in 10 universal culture media (Table 6), referring to the methods of Ruan Jisheng (1977), Xu Lihua and Li Wenjun (2007), Ruan Jisheng and Huang Ying (2010), etc. The physiological and biochemical characteristics of the strain, such as carbon and nitrogen source utilization, starch hydrolysis, cellulose enzymatic hydrolysis, gelatin liquefaction, milk coagulation and peptone production, hydrogen sulfide production, salt tolerance, and growth temperature, were observed.

[0067] 3.1.5 Molecular identification DNA extraction, molecular amplification, and sequencing of 16S rDNA of strain HMQAUfx21024 were performed according to the method of Liang Chunhao in Chinese patent CN 114874948 B (a strain of Bacillus subtilis HMQAU20091, a bacterial agent, a preparation method, and an application thereof) (Liang Chunhao, 2016). The sequencing results were used to construct a phylogenetic tree using MEGA11.

[0068] 3.2 Results and Analysis 3.2.1 Morphology and culture characteristics Morphological characteristics: The strain HMQAUfx21024 was cultured on Gao's No. 1 plate at 28℃ for 5 days and then observed. Figure 3 and Figure 4As shown. Its colony is round with concentric rings around it, and the hyphae are radial; the hyphae in the base have no septa, are well developed, and are pink; the aerial hyphae grow luxuriantly, and are white or mouse-gray; the spores are spherical or elliptical, with a smooth surface, and the spores are spiral; no pigment is produced; it has typical Streptomyces characteristics.

[0069] Culture characteristics: The culture characteristics of strain HMQAUfx21024 on the six culture media described in 3.1.2 are shown in Table 6 and Figure 5 As shown. A rich amount of aerial hyphae can be produced on ISP2, Gao's agar No. 1, and PDA medium, but less on ISP3, ISP4, and ISP5; the base hyphae do not produce soluble pigments on the six culture media, and appear pink in ISP2, ISP3, ISP5, and Gao's agar No. 1, with slightly different shades of color. The base hyphae on PDA are goose yellow, but less in ISP4 and white.

[0070] Table 6 Culture characteristics of strain HMQAUfx21024 on different culture media

[0071] 3.2.2 Physiological and biochemical characteristics The results of physiological and biochemical tests are shown in Table 7. The strain HMQAUfx21024 cannot grow normally when the NaCl concentration is >6%, but grows well when it is <4%; it can grow at temperatures between 10 and 40°C, with 28°C being the optimal growth temperature; the reactions of gelatin liquefaction, milk coagulation and peptization, starch hydrolysis, and cellulose enzymatic hydrolysis are positive; hydrogen sulfide production is negative; except for mannitol, glucose, fructose, maltose, galactose, and sucrose can be used as the only carbon sources for this strain; urea, diammonium phosphate, beef extract, peptone, trypsin, and yeast powder can be used as the only nitrogen sources for this strain; but it cannot utilize ammonium nitrate, ammonium chloride, and glutamic acid. Based on the culture characteristics and physiological and biochemical characteristics of the strain HMQAUfx21024, the strain was preliminarily identified as Streptomyces ( Streptomyces ).

[0072] Table 7 Physiological and biochemical characteristics of strain HMQAUfx21024

[0073] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0074] 3.2.3 Molecular identification After PCR amplification of the 16S rDNA sequence of strain HMQAUfx21024, a clear band of about 1500 bp in length was obtained by electrophoresis; bidirectional sequencing was performed by Qingke Biotechnology Co., Ltd., and the sequence length of the strain was determined to be 1486 bp.

[0075] The strain HMQAUfx21024 sequence was submitted to the NCBI gene database to search for 16S rDNA sequences of related strains and construct a phylogenetic tree. Figure 6 As shown in the figure, strain HMQAUfx21024 was clustered with strains with sequence numbers KY412831, NR041186, MH432682, and HQ850417. Combining the morphological characteristics, physiological and biochemical characteristics, and molecular identification results of the strain, strain HMQAUfx21024 was finally identified as Streptomyces griseoaurantifolia ( Streptomyces griseoaurantiacus ).

[0076] Example 4 Inhibitory effect of strain HMQAUfx21024 on different pathogens 4.1 Test plant diseases and their pathogens Lily Gray Mold: Botrytis cinerea Botrytis cinerea HMQAU200037; Bean gray mold: Botrytis cinerea Botrytis cinerea HMQAU170042; Celery Sclerotinia: Sclerotinia sclerotiorum Sclerotinia sclerotia HMQAU170216; Chinese cabbage sclerotinia disease: Sclerotinia sclerotiorum Sclerotinia sclerotia HMQAU180110; Grape white rot: Aspergillus niger Coniella diplodiella HMQAU170082; Blueberry Lasiospora branch blight: Pseudococcus spp. Lasiodiplodia pseudotheobromae HMQAU140073; Spinach Pythium root rot: Pythium terrestris Pythium irregulare HMQAU210091; Pythium arboreatum Pythium forest HMQAU210092; Lettuce leaf spot: Sphaerotheca oleracea Paper mulberry HMQAU210122, Stolonifera Stemphylium botryosum HMQAU210121; Fusarium root rot of spinach: Fusarium oxysporum Fusarium oxysporum HMQAU170173; Blueberry leaf spot: Pseudomonas Pestalotiopsis sp. HMQAU180119; Phomopsis Phomopsis sp. HMQAU210069; Quinoa branch blight: Botrytis cinerea Botryosphaeria dothidae HMQAU190265; Siamese Anthrax Colletotrichum siamense HMQAU190267, Stemomyces betae Beetroot soup HMQAU190266; Garlic dry rot: Fusarium oxysporum Fusarium oxysporum HMQAU210030, Fusarium solani Fusarium solani HMQAU210027; Kiwifruit soft rot: Mucor circinelloides I am sick. circinelloid HMQAU150050; Wheat stem rot: Pseudomonas graminearum Fusarium pseudograsses HMQAU220027, all provided by the Mycology Laboratory of Qingdao Agricultural University.

[0077] 4.2 Test methods The mycelium growth rate method (Chen Nianchun, 1991) was used to mix the fermentation filtrate of HMQAUfx21024 with the melted and cooled PDA culture medium (45-50℃) (diluted 10 times) and pour it into a plate. The PDA culture medium with an equal volume of Gao's No. 1 liquid culture medium was added as the control (diluted 10 times). A pathogen cake with a diameter of 5 mm was inoculated in the center of the plate. When the pathogen colony in the control group filled 3 / 4 of the culture dish, the colony radius of the control group and the treatment group was measured by the cross method, and the inhibition rate was calculated. Three replicates were set for each treatment.

[0078] Inhibition rate (%) = [(control colony radius (mm) - treatment colony radius (mm)) / control colony radius (mm)] × 100% 4.3 Inhibitory effects of Streptomyces griseoaurantifolia HMQAUfx21024 on different pathogens The test results of the mycelium growth rate method are shown in Table 8. The fermentation filtrate has different degrees of inhibitory effect on 20 pathogens. Among them, it has obvious inhibitory effect on Mucor circinelloides, Pythium arborescens, Botrytis cinerea, and Pseudococcidioides spp., with an inhibition rate of more than 95%; followed by Pythium sclerotiorum, Sclerotinia sclerotiorum, Botrytis cinerea, Phomopsis spp., Pseudococcidioides spp., and Pseudococcidioides spp., with inhibition rates of more than 75%; the inhibition rates of the other 8 pathogens are also between 30.30% and 64.55%. It shows that the strain HMQAUfx21024 has a wide antibacterial spectrum, good antibacterial effect, and high biocontrol potential. The difference significance analysis shows that the inhibitory effect of the fermentation filtrate of the strain on different pathogens is significantly different.

[0079]

[0080] Note: The letters after the numbers in the same column indicate P <0.05 level, significant difference (Duncan's new multiple range method) Example 5 The confrontation effect of strain HMQAUfx21024 on different pathogens 5.1 Test plant diseases and their pathogens Pepper blight: Phytophthora capsici Phytophthora capsicum HMQAU240336; Bean gray mold: Botrytis cinerea Botrytis cinerea HMQAU170042; Ginger root rot: Pythium glomerulosa Pythium myriotylumHMQAU240324; Rhizoctonia solani root rot of Chinese cabbage: Rhizoctonia solani Rhizoctonia solani HMQAU230017; Tomato leaf mold: Cladosporium fulva Cladosporium fulvum HMQAU240001; Solanum early blight: Alternaria solani Alternaria solani HMQAU230064; Garlic dry rot: Fusarium solani Fusarium solani HMQAU210027; Cucumber rot: Pythium aphanidermatum Pythium aphaniderm HMQAU240161; Strawberry Anthracnose: Colletotrichum Colletorichum gloeosporioides HMQAU240135; Cucumber Pythium root rot: Pythium spinulosum Pythium spinosum HMQAU230410; Cucumber wilt: Fusarium oxysporum Fusarium oxysporum HMQAU23413; Watermelon wilt: Fusarium oxysporum Fusarium oxysporum HMQAU24005; Cucumber Anthracnose: Colletotrichum rotundifolia Colletotrichum orbiculare HMQA U240368, all provided by the Mycology Laboratory of Qingdao Agricultural University.

[0081] 5.2 Test methods Plate confrontation method: streak inoculate Streptomyces griseo-orange in the middle of a 9 cm diameter PDA plate Streptomyces gray-orange HMQAUfx21024, symmetrically inoculate the same pathogenic bacteria lawn 2.25 cm away from the strain line, and use the pathogenic bacteria plate without actinomycetes as the control group. Culture at 28°C until the control colony area occupies 3 / 4 of the plate, measure the colony radius, and calculate the inhibition rate. The inhibition rate = [(colony radius of the control group - colony radius of the treatment group) / colony radius of the control group] × 100% (Wang Qing et al., 2013).

[0082] 5.3 Inhibitory effects of Streptomyces griseo-orange HMQAUfx21024 on different pathogens The test results of the plate confrontation method are shown in Table 9. The fermentation filtrate has different degrees of inhibitory effect on 13 pathogens. Among them, the inhibition rate of pepper phytophthora reached 91.43%; followed by Botrytis cinerea and Pythium spp., with inhibition rates of more than 85%; the inhibition rates of 7 pathogens such as Rhizoctonia solani were also between 48.57% and 71.43%; the inhibition effect on Fusarium oxysporum and Colletotrichum spp. was poor, between 5.00% and 25.00%. This shows that the strain HMQAUfx21024 has a broad antibacterial spectrum, good antibacterial effect, and high biocontrol potential. The significant difference analysis showed that the inhibitory effect of the strain on different pathogens was significantly different, such as Figure 7 shown.

[0083]

[0084] It should be pointed out that the specific implementation methods are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-mentioned embodiments, and there are many variations. Those skilled in the art who can obtain the invention clearly disclosed in the present invention or without objection based on the written description of the document should be considered as the scope of protection of this patent.

Claims

1. A strain of Streptomyces griseo-orange ( Streptomyces griseoaurantiacus ) strain HMQAUfx21024, characterized in that The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 3, 2025, with the deposit registration number: CGMCC No. 33290.

2. A biocontrol agent, characterized in that: The active ingredient of the biocontrol agent includes the bacterial suspension, fermentation liquid or fermentation filtrate of the Streptomyces griseoaurantifolia strain HMQAUfx21024 as claimed in claim 1.

3. The biocontrol agent according to claim 2, characterized in that The total live bacterial concentration of Streptomyces griseo-orange strain HMQAUfx21024 in the biocontrol agent is 1×10 5 ~2×10 5 cfu / mL.

4. The method for preparing the biocontrol agent according to claim 2, characterized in that: The specific steps include: The Streptomyces griseo-orange strain HMQAUfx21024 was activated on Gao's No. 1 solid culture medium at 25-35°C for 3-5 days, and then sterile water was added to prepare a spore suspension to obtain a biocontrol agent.

5. The method for preparing the biocontrol agent according to claim 2, characterized in that: The specific steps include: S1. Cultivation of seed solution: After activating the Streptomyces griseo-orange strain HMQAUfx21024 on Gao's solid medium No. 1 at 25-35°C for 3-5 days, 3-5 bacterial cakes were taken from the edge of the colony and inoculated into Gao's liquid medium No. 1 at 25-35°C and 150-200 rpm for 3-5 days to obtain seed solution; S2. Preparation of fermentation broth: The seed solution obtained in step S1 was added to a conical flask containing a certain amount of basic fermentation medium at a ratio of 10 to 15% inoculation amount, and cultured at 25 to 35 ° C and 150 to 220 rpm with shaking for 2 to 5 days to prepare a fermentation broth to obtain a biocontrol agent.

6. The method for preparing the biocontrol agent according to claim 2, characterized in that: The specific steps include: S1. Cultivation of seed solution: After activating the Streptomyces griseo-orange strain HMQAUfx21024 on Gao's solid medium No. 1 at 25-35°C for 3-5 days, 3-5 bacterial cakes were taken from the edge of the colony and inoculated into Gao's liquid medium No. 1 at 25-35°C and 150-200 rpm for 3-5 days to obtain seed solution; S2. Preparation of fermentation broth: The seed solution obtained in step S1 was added to a certain amount of the base fermentation medium in an inoculum ratio of 10 to 15%, and the fermentation broth was prepared by shaking and culturing at 25 to 35 ° C and 150 to 220 rpm for 2 to 5 days; S3. Preparation of fermentation filtrate: The fermentation broth obtained from step S2 is centrifuged at 4-6°C, 6000-9000rpm for 20-30min, pH 7-8, and filtered through a microporous membrane to prepare a fermentation filtrate, that is, a biocontrol agent.

7. The method for preparing the biocontrol agent according to claim 6, characterized in that: The components and contents of Gao's No. 1 solid culture medium in step S1 are as follows: 20g soluble starch, 0.5g sodium chloride, 1g potassium nitrate, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferric sulfate, 20g agar, and 1000mL distilled water; the components and contents of Gao's No. 1 liquid culture medium in step S1 are as follows: 20g soluble starch, 0.5g sodium chloride, 1g potassium nitrate, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferric sulfate, and 1000mL distilled water; the basic fermentation medium in step S2 is millet medium, and the components and contents are as follows: 10.0g millet, 10.0g glucose, 3.0g peptone, 2.5g sodium chloride, 2.0g calcium carbonate, 1000mL distilled water, pH7.2-7.

4.

8. Use of the Streptomyces griseo-orange strain HMQAUfx21024 according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 7 in the following (a1) or (a2) or (a3) ​​or (a4), characterized in that: (a1) inhibiting plant pathogens; (a2) preparing products for inhibiting plant pathogens; (a3) ​​preventing and controlling diseases caused by plant pathogens; (a4) preparing products for preventing and controlling diseases caused by plant pathogens; the plant pathogen is Pseudocerona cubaniensis ( Pseudoperonospora cubensis )、Spores of sphaerocephala ( Corynespora cassicola )、Mucor circinelloides( Mucor circinelloides )、Forest-dwelling Pythium Pythium sylvaticum )、Botrytis cinerea( Botryosphaeria dothidea )、False cocoa hair color two cells ( Lasiodiplodia pseudotheobromae )、Pythium terrestris( Pythium irregulare ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum )、Botrytis cinerea( Botrytis cinerea )、Phomopsis sphaeroides( Phomopsis sp.), Pseudodiscus hirsutus ( Pestalotiopsis sp.), Paper-like spores ( Ulocladium chartarum )、Stoma betae Phoma betae )、Fusarium solani( Fusarium solani ), Pseudofusorium graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Siamese Anthrax( Colletotrichum siamense )、White rot spores( Coniella diplodiella )、Stolonifera( Stemphylium botryosum )、Phytophthora capsici( Phytophthora capsici )、Pythium glomerulosa( Pythium myriotylum )、Rhizoctonia solani( Rhizoctonia solani )、Clondrosporium fulva Cladosporium fulvum ) Alternaria solani )、Pythium aphanidermatum( Pythium aphanidermatum )、Collegiosporium Colletorichum gloeosporioides )、Pythium spinulosum( Pythium spinosum )、Circular Sporangium ( Colletotrichum orbiculare ).

9. Use of the Streptomyces griseo-orange strain HMQAUfx21024 according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 7 in plant disease control, characterized in that: The plant disease is any one of cucumber downy mildew, cucumber target spot, lily gray mold, bean gray mold, celery sclerotinia, Chinese cabbage sclerotinia, grape white rot, blueberry hairy dispora branch blight, spinach rot, lettuce leaf spot, spinach Fusarium root rot, blueberry leaf spot, quinoa branch blight, garlic dry rot, kiwi soft rot, wheat stem rot, pepper blight, ginger root rot, cabbage Rhizoctonia root rot, tomato leaf mold, eggplant early blight, cucumber cotton rot, strawberry anthracnose, cucumber rot, cucumber wilt, watermelon wilt, and cucumber anthracnose.

10. A method for preventing and controlling plant diseases, characterized in that: When preventing and controlling plant diseases, the griseo-orange Streptomyces strain HMQAUfx21024 according to claim 1, the biocontrol agent according to any one of claims 2 to 3, or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 7 is coated, dipped, sprayed, atomized, irrigated, powdered, broadcasted, foamed, applied or sprayed on the disease-prone parts of the plant.

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